WO1997020046A1 - Molecule d'adn, antigene tn et molecule d'adhesion appartenant a la superfamille des immunoglobulines - Google Patents

Molecule d'adn, antigene tn et molecule d'adhesion appartenant a la superfamille des immunoglobulines Download PDF

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Publication number
WO1997020046A1
WO1997020046A1 PCT/US1996/018551 US9618551W WO9720046A1 WO 1997020046 A1 WO1997020046 A1 WO 1997020046A1 US 9618551 W US9618551 W US 9618551W WO 9720046 A1 WO9720046 A1 WO 9720046A1
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dnaml
protein
antigen
cells
antibody
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PCT/US1996/018551
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English (en)
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Akira Shibuya
Lewis L. Lanier
Joseph H. Phillips, Jr.
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Schering Corporation
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Priority to AU77396/96A priority Critical patent/AU7739696A/en
Publication of WO1997020046A1 publication Critical patent/WO1997020046A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily

Definitions

  • DNAM a NK antigen and adhesion molecule of the immunoglobulin superfamily
  • the immune system of vertebrates consists of a number of organs and several different cell types including monocytes, granulocytes, and
  • lymphoid cell lineage 1 5 lymphocytes.
  • B cells which were originally characterized as differentiating in fetal liver or adult bone marrow
  • T cells which were originally characterized as differentiating in the thymus
  • NK cells natural killer cells See, e.g., Paul (ed.) (1989) Fundamental Immunology (2d ed.) Raven Press, New York. 0
  • soluble proteins e.g., cytokines
  • cell surface antigens e.g., CD markers
  • the present invention is based, in part, upon the discovery of a cDNA clone encoding a member of a family of cell surface markers, initially characterized on natural killer (NK) and T cells and designated DNAX Accessory Molecule 1 (DNAM1). Interference of DNAM1 interaction with a target cell can affect cytotoxic killing activity and sometimes cytokine production.
  • the invention embraces isolated genes encoding the proteins of the invention, variants of the encoded proteins, e.g., mutations (muteins) of the natural sequence, species and allelic variants, fusion proteins, chemical mimetics, antibodies, and other structural or functional analogues. Various uses of these different nucleic acid or protein compositions are also provided.
  • the present invention provides nucleic acids encoding a DNAM1 protein or fragment thereof; a substantially pure DNAM1 or peptide thereof, or a fusion protein comprising DNAM1 sequence; and an antibody raised to a DNAM1 protein.
  • the nucleic acid can comprise a sequence of SEQ ID NO: 1.
  • the protein or peptide can be from a primate, including a human; comprise at least one polypeptide segment of SEQ ID NO: 2; or exhibit a post-translational modification pattern distinct from natural DNAMl protein.
  • the protein or peptide further exhibits exhibits properties of Table 2.
  • a further embodiment is a composition comprising such a protein and a pharmaceutically acceptable carrier.
  • the antigen can be a primate protein, including a human; the antibody is raised against a protein sequence of SEQ ID NO: 2; the antibody is a monoclonal antibody; or the antibody is labeled.
  • the invention also embraces a kit comprising a substantially pure nucleic acid encoding a DNAMl protein or peptide; a substantially pure DNAMl protein or fragment, e.g., as a positive control; or an antibody or receptor which specifically binds a DNAMl protein.
  • reagents also provides methods of developing agonists or antagonists of a DNAMl protein which can modulate DNAMl -dependent immune responses.
  • the antagonist might be an antibody against a mammalian DNAMl protein; or the cell may be a hematopoietic cell, including a lymphoid cell.
  • Such a reagent may be combined with another costimulatory reagent, e.g., CD18, CDla ligand (anti-LFA-1), and/or anti- CD2.
  • the present invention provides DNA sequence encoding various mammalian proteins expressed on human NK cells, T cells, monocytes, and subsets of B cells.
  • the cDNA sequence exhibits various features which are characteristic of mRNAs encoding physiologically and /or developmentally important cell markers. See, e.g., Yokoyama (1993) Ann. Rev. Immunol. 11:613-35.
  • the human gene described herein contains an open reading frame encoding a presumptive 336 amino acid protein.
  • DNAMl proteins The natural proteins should be capable of modulating or regulating cytotoxic killing or cytokine production by T cells or NK cells. Initial studies had localized this protein to various hematopoietic cell types. See, e.g., Table 1. Biochemical properties are described in Table 2.
  • HeLa epithelial
  • PA-1 (ovarian teratocarcinoma) Table 2: Biochemical Properties of DNAMl markers.
  • DNAM-1 molecule is a signal transducing accessory molecule which plays a major role in regulating cell-cell interactions during various immune responses.
  • combination therapy with other accessory molecules should be useful. See, e.g., Weiss (1994) in Paul (ed.) Fundamental Immunology (3d ed.) Raven Press, N.Y. These responses include those where T-cell activation or NK cell activation occur, e.g, in activated killing.
  • Stimulation of the accessory molecule should allow activation to increase targeted killing and possibly cytokine production. In other situations, blocking of the reagents may suppress the normal activation of targeted killing or other local inflammation or infections.
  • the DNAMl signal pathway may be especially important in those individuals.
  • SEQ ID NO: 1 discloses the nucleotide and amino acid sequences of one protein of the DNAMl family.
  • the described nucleotide sequence, fragments, and related reagents can be employed in constructing a DNA clone useful for expressing DNAMl protein, or, e.g., isolating a similar gene from another natural source, including other members of the family.
  • the sequences will be useful in isolating other genes, e.g., allelic variants or alternatively spliced isoforms, from human.
  • the purified protein or defined peptides are useful for generating antibodies by standard methods, as described below. Synthetic peptides or purified protein can be presented to an immune system to generate a specific binding composition, e.g., monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology
  • the specific binding composition can be used for screening of an expression library made from a cell line which expresses a DNAMl protein.
  • the screening can be standard staining of surface expressed protein, or by panning. Screening of intracellular expression can also be performed by various staining or immunofiuorescence procedures.
  • the binding compositions can also be used to affinity purify or sort out cells expressing the protein.
  • This invention contemplates use of isolated DNA or fragments to encode a biologically active DNAMl protein or polypeptide.
  • this invention covers isolated or recombinant DNA which encodes a biologically active protein or polypeptide and which is capable of hybridizing under appropriate conditions with the DNA sequences described herein.
  • Said biologically active protein or polypeptide can be an intact antigen, or fragment, and have an amino acid sequence as disclosed in SEQ ID NO: 2.
  • this invention covers the use of isolated or recombinant DNA, or fragments thereof, which encode proteins which share epitopes or biological properties of a DNAMl protein or which were isolated using cDNA encoding a DNAMl protein as a probe.
  • the isolated DNA can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others.
  • nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from at least some other components which naturally accompany a native sequence, e.g., ribosomes, polymerases, and /or flanking genomic sequences from the originating species.
  • the term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogues biologically synthesized by heterologous systems.
  • An isolated form of the molecule includes substantially pure proteins or peptides. Alternatively, a purified species may be separated from host components from a recombinant expression system.
  • An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity. Some variability in post-translational modification may exist, e.g., some glycosylation variation.
  • a "recombinant" nucleic acid is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants.
  • products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oligonucleotide process. Such is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms. Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • site specific targets e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • a similar concept is intended for a recombinant, e.g., fusion, polypeptide.
  • synthetic nucleic acids which, by genetic code redundancy, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various different species variants.
  • a significant "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides.
  • a DNA which codes for a DNAMl protein will be particularly useful to identify genes, mRNA, and cDNA species which code for related or homologous proteins, as well as DNAs which code for similar proteins. There are likely similar proteins in other primates. Various DNAMl proteins should exhibit high identity and are encompassed herein. However, even proteins that have a more distant evolutionary relationship to the antigen can readily be isolated under appropriate conditions using these sequences if they are sufficiently identical. Primate DNAMl proteins are of particular interest.
  • This invention further covers recombinant DNA molecules and fragments having a DNA sequence identical to or highly similar to the isolated DNAs set forth herein.
  • the sequences will often be operably linked to DNA segments which control transcription, translation, and DNA replication.
  • recombinant clones derived from the genomic sequences e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow (1992) "Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn et al.
  • nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least about 65%, often at least 80%, preferably at least about 90%, more preferably at least about 95 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides.
  • segment will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence derived from SEQ ID NO: 1.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, preferably at least about 65%, more preferably at least about 75%, and most preferably at least about 90%. See, Kanehisa (1984) Nuc. Acids Res. 12:203-213.
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides.
  • Stringent conditions in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters, typically those controlled in hybridization reactions.
  • Stringent temperature conditions will usually include temperatures in excess of about 30° C to about 70° C.
  • Stringent salt conditions will ordinarily be less than about 1000 mM, preferably less than about 150 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson (1968) T. Mol. Biol. 31:349- 370.
  • DNAMl The predicted sequence of human DNAMl amino acid sequence is shown in SEQ ID NO: 2.
  • the peptide sequences allow preparation of peptides to generate antibodies to recognize such segments,
  • DNAMl shall encompass, when used in a protein context, a protein having an amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.
  • the present invention also encompasses a primate, e.g., human, derived polypeptide which exhibits similar biological function or interacts with DNAMl protein specific binding components.
  • binding components typically bind to a DNAMl protein with high affinity, e.g., at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM.
  • polypeptide includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least 8 amino acids.
  • Substantially pure in the polypeptide context, typically means that the protein is free from other contaminating proteins, nucleic acids, and other biologicals derived from the original source organism. Purity may be assayed by standard methods, and will ordinarily be at least about 40% pure. Preferably the purity of the purity ranges from 90% to 99%+. The analysis may be weight or molar percentages, evaluated, e.g., by gel staining, spectrophotometry, or terminus labeling.
  • a binding composition refers to molecules that bind with specificity to DNAMl protein, e.g., in an antibody-antigen interaction.
  • the invention also encompasses interaction in, e.g. a ligand-receptor type fashion, or compounds, e.g., proteins which specifically associate with DNAMl protein, e.g., in a natural physiologically relevant protein- protein interaction, either covalent or non-covalent.
  • the molecule may be a polymer, or chemical reagent. The interaction should exhibit specific affinity.
  • a functional analog may be a protein with structural modifications, or may be a wholly unrelated molecule, e.g., which has a molecular shape which interacts with the appropriate binding determinants.
  • the proteins may serve as agonists or antagonists of a receptor, see, e.g., Goodman et al. (eds.) (1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed.) Pergamon Press.
  • Solubility of a polypeptide or fragment depends upon the environment and the polypeptide. Many parameters affect polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent. Typically, the temperature at which the polypeptide is used ranges from about 4° C to about 65° C. Usually the temperature at use is greater than about 18° C and more usually greater than about 22° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37° C for humans, though under certain situations the temperature may be raised or lowered in situ or in vitro.
  • the electrolytes will usually approximate in situ physiological conditions, but may be modified to higher or lower ionic strength where advantageous.
  • the actual ions may be modified, e.g., to conform to standard buffers used in physiological or analytical contexts.
  • the size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state.
  • the polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubility, or associated with lipids or detergents in a manner which approximates natural lipid bilayer interactions.
  • the solvent will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological solvent.
  • the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5.
  • a detergent will be added, typically a mild non- denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3-([3- cholamidopropyl]dimethylammonio)-l-propane sulfonate), or a low enough concentration as to avoid significant disruption of structural or physiological properties of the antigen.
  • Solubility is reflected by sedimentation measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule under particular conditions.
  • the determination of the sedimentation velocity was classically performed in an analytical ultracentrifuge, but is typically now performed in a standard ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d ed.), W.H. Freeman; and Cantor and Schimmel (1980) Biophysical Chemistry, parts 1-3, W.H. Freeman & Co., San Francisco, CA.
  • a sample containing a putatively soluble polypeptide is spun in a standard full sized ultracentrifuge at about 50K rpm for about 10 minutes, and soluble molecules will remain in the supernatant.
  • a soluble particle or polypeptide will typically be less than about 30S.
  • DNA which encodes the DNAMl protein or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples.
  • This DNA can be expressed in a wide variety of host cells for the synthesis of a full-length protein or fragments which can in turn, for example, be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified molecules; and for structure /function studies.
  • Each antigen or its fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be substantially purified to be free of protein or cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and /or diluent.
  • the antigen, or portions thereof, may be expressed as fusions with other proteins.
  • Expression vectors are typically self-replicating DNA or RNA constructs containing the desired antigen gene or its fragments, usually operably linked to suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression within a suitable host. The specific type of control elements necessary to effect expression will depend upon the eventual host cell used.
  • the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation.
  • Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell.
  • the vectors of this invention contain DNA which encodes a DNAMl protein, or a fragment thereof, typically encoding a biologically active polypeptide.
  • the DNA can be under the control of a viral promoter and can encode a selection marker.
  • This invention further contemplates use of such expression vectors which are capable of expressing eukaryotic cDNA coding for a DNAMl protein in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the eukaryotic cDNA coding for the antigen is inserted into the vector such that growth of the host containing the vector expresses the cDNA in question.
  • expression vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, e.g., it is possible to effect transient expression of the antigen or its fragments in various hosts using vectors that do not contain a replication origin that is recognized by the host cell. It is also possible to use vectors that cause integration of a DNAMl gene or its fragments into the host DNA by recombination, or to integrate a promoter which controls expression of an endogenous gene.
  • Vectors as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host.
  • Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, e.g., Pouwels et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual, Elsevier, N.Y., and Rodriquez et al. (1988)(eds.) Vectors: A Survey of Molecular Cloning Vectors and Their Uses, Buttersworth, Boston, MA.
  • Transformed cells include cells, preferably mammalian, that have been transformed or transfected with vectors containing a DNAMl gene, typically constructed using recombinant DNA techniques.
  • Transformed host cells usually express the antigen or its fragments, but for purposes of cloning, amplifying, and manipulating its DNA, do not need to express the protein.
  • This invention further contemplates culturing transformed cells in a nutrient medium, thus permitting the protein to accumulate in the culture. The protein can be recovered, either from the culture or from the culture medium.
  • DNA sequences are operably linked when they are functionally related to each other.
  • DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation.
  • operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression.
  • Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes.
  • Prokaryotes include both gram negative and gram positive organisms, e.g., E. coli and B. subtilis.
  • Lower eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and species of the genus Dictyostelium.
  • Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents.
  • Prokaryotic host-vector systems include a wide variety of vectors for many different species. As used herein, E. coli and its vectors will be used generically to include equivalent vectors used in other prokaryotes.
  • a representative vector for amplifying DNA is pBR322 or many of its derivatives.
  • Vectors that can be used to express the DNAMl proteins or its fragments include, but are not limited to, such vectors as those containing the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybrid promoters such as ptac (pDR540). See Brosius et al.
  • Lower eukaryotes e.g., yeasts and Dictyostelium, may be transformed with vectors encoding DNAMl proteins.
  • the most common lower eukaryotic host is the baker's yeast, Saccharomyces cerevisiae. It will be used to generically represent lower eukaryotes although a number of other strains and species are also available.
  • Yeast vectors typically consist of a replication origin (unless of the integrating type), a selection gene, a promoter, DNA encoding the desired protein or its fragments, and sequences for translation termination, polyadenylation, and transcription termination.
  • Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or such inducible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter.
  • Suitable vectors include derivatives of the following types: self- replicating low copy number (such as the YRp-series), self-replicating high copy number (such as the YEp-series); integrating types (such as the Yip-series), or mini-chromosomes (such as the YCp-series).
  • Higher eukaryotic tissue culture cells are the preferred host cells for expression of the functionally active DNAMl protein.
  • many higher eukaryotic tissue culture cell lines are workable, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source.
  • mammalian cells are preferred, in that the processing, both cotranslationally and posttranslationally. Transformation or transfection and propagation of such cells has become a routine procedure.
  • useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS) cell lines.
  • Expression vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as from adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus. Representative examples of suitable expression vectors include pCDNAl; pCD, see Okayama et al. (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas et al. (1987) £ell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
  • DNAMl protein polypeptide in a system which provides a specific or defined glycosylation pattern.
  • the usual pattern will be that provided naturally by the expression system.
  • the pattern will be modifiable by exposing the polypeptide, e.g., an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system.
  • the DNAMl protein gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable or approximated in prokaryote or other cells. Cell lines for expression may be selected, e.g., based upon desired glycosylation patterns.
  • the DNAMl protein may be engineered to be phosphatidyl inositol (PI) linked to a cell membrane, but can be removed from membranes by treatment with a phosphatidyl inositol - cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C.
  • PI phosphatidyl inositol
  • a phosphatidyl inositol - cleaving enzyme e.g., phosphatidyl inositol phospholipase-C.
  • a soluble version may result from cleavage at a residue near the PI linkage.
  • DNAMl protein has been characterized, fragments or derivatives thereof can be prepared by conventional processes for synthesizing peptides. These include processes such as are described in Stewart and Young (1984) Solid Phase Peptide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky and Bodanszky (1984) The Practice of Peptide Synthesis, Springer-Verlag, New York; and
  • an azide process for example, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative- reductive process, or a dicyclohexylcarbodiimide (DCCD) /additive process can be used.
  • Solid phase and solution phase syntheses are both applicable to the foregoing processes.
  • the DNAMl protein, fragments, or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. Amino groups that are not being used in the coupling reaction are typically protected to prevent coupling at an incorrect location.
  • the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group.
  • the insoluble carrier is not particularly limited as long as it has a bmding capability to a reactive carboxyl group.
  • examples of such insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin, hydroxymethyl resins, phenol resins, tert- alkyloxycarbonyl-hydrazidated resins, and the like.
  • An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize the peptide step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide. This solid-phase approach is generally described by Merrifield et al. (1963) in I. Am. Chem. Soc. 85:2149-2156.
  • the prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, for example, by extraction, precipitation, electrophoresis and various forms of chromatography, and the like.
  • the DNAMl proteins of this invention can be obtained in varying degrees of purity depending upon its desired use. Purification can be accomplished by use of the protein purification techniques disclosed herein or by the use of the antibodies herein described in immunoabsorbant affinity chromatography.
  • This immunoabsorbant affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate source cells, lysates of other cells expressing the protein, or lysates or supematants of cells producing the DNAMl protein as a result of DNA techniques, see below.
  • This invention also provides proteins or peptides having substantial amino acid sequence homology with the amino acid sequence of the DNAMl protein.
  • the variants include species and allelic variants. Amino acid sequence homology, or sequence identity, as determined by optimizing residue matches, if necessary, by introducing gaps as required. This changes when considering conservative substitutions as matches. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • homologous amino acid sequences are typically intended to include natural allelic and interspecies variations in each respective protein sequence. Typical homologous proteins or peptides will have from 25-100% homology (if gaps can be introduced), to 50-100% homology (if conservative substitutions are included) with the amino acid sequence of the DNAMl protein. Identity measures will be at least about 35%, and preferably at least about 80%. See also Needleham et al. (1970) I. Mol. Biol. 48:443-453; Sankoff et al.
  • the isolated DNA encoding a DNAMl protein can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode these antigens, their derivatives, or proteins having similar physiological, immunogenic, or antigenic activity. These modified sequences can be used to produce mutant antigens or to enhance expression.
  • mutant DNAMl protein derivatives include predetermined or site- specific mutations of the respective protein or its fragments.
  • mutant DNAMl protein encompasses a polypeptide otherwise falling within the homology definition of the human DNAMl protein as set forth above, but having an amino acid sequence which differs from that of DNAMl protein as found in nature, whether by way of deletion, substitution, or insertion.
  • site specific mutant DNAMl protein generally includes proteins having significant homology with a protein having sequences of SEQ ID NO: 1, and as sharing various biological activities, e.g., antigenic or immunogenic, with those sequences, and in preferred embodiments contain most of the disclosed sequences. Similar concepts apply to different DNAMl proteins, particularly those found in various mammals, e.g., primates, including human. As stated before, it is emphasized that descriptions are generally meant to encompass all DNAMl proteins, not limited to the specific embodiment discussed.
  • DNAMl protein mutagenesis can be conducted by making amino acid insertions or deletions. Substitutions, deletions, insertions, or any combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, e.g., by M13 primer mutagenesis or polymerase chain reaction (PCR) techniques. See also Sambrook et al. (1989) and Ausubel et al. (1987 and Supplements). The mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins.
  • a heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner.
  • the fusion product of an immunoglobulin with a DNAMl polypeptide is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties derived from each source peptide.
  • a similar concept applies to heterologous nucleic acid sequences.
  • new constructs may be made from combining similar functional domains from other proteins. For example, antigen-binding or other segments may be "swapped" between different new fusion polypeptides or fragments.
  • a double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence, e.g., PCR techniques.
  • the blocking of physiological response to DNAMl proteins may result from the inhibition of binding of the antigen to its natural binding partner, e.g., through competitive inhibition.
  • in vitro assays of the present invention will often use isolated protein, membranes from cells expressing a recombinant membrane associated DNAMl protein, soluble fragments comprising binding segments, or fragments attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or protein mutations and modifications, e.g., analogues.
  • This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to antigen or binding partner fragments compete with a test compound for binding to the protein.
  • the antibodies can be used to detect the presence of any polypeptide which shares one or more antigenic binding sites of the protein and can also be used to occupy binding sites on the protein that might otherwise interact with a binding partner.
  • neutralizing antibodies against the DNAMl protein and soluble fragments of the antigen which contain a high affinity receptor binding site can be used to inhibit antigen function in tissues, e.g., tissues experiencing abnormal physiology.
  • certain antibodies, e.g., antagonists, which bind DNAMl can activate the NK or cytotoxic T cell to kill a target which otherwise would not be killed.
  • the DNAMl may be an important positive regulator of cytotoxic physiology.
  • it may be useful to target undesirable cells, e.g., tumor cells, to express such an antibody, antagonist, or binding partner of DNAMl.
  • an antibody may be useful in blocking target cell killing by an NK cell or T cell, or monocyte.
  • Antibodies against the DNAMl protein can modulate NK cell- and antigen specific T cell- mediated cytotoxicity. Moreover, these antibodies can, either directly or indirectly, regulate cytokine production by the cells during immune responses. These include effects on IFN- ⁇ , TNF- ⁇ , and GM-CSF production.
  • DNAMl antibodies are variable, depending, e.g., upon the target cell HLA genotype, but its effect is on non-specific NK cell activity, and upon antigen-specific T cell cytotoxicity. Moreover, the DNAMl has effects, either direct or indirect, upon cytokine production by the cells. These include effects on IFN- ⁇ and TNF- ⁇ .
  • Derivatives of the DNAMl antigens include amino acid sequence mutants, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties.
  • Covalent derivatives can be prepared by linkage of functionalities to groups which are found in the DNAM1 amino acid side chains or at the N- or C- termini, by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group containing residues, e.g., lysine or arginine.
  • Acyl groups are selected from the group of alkyl- moieties including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species. Covalent attachment to carrier proteins may be important when immunogenic moieties are haptens.
  • glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are by exposing the polypeptide to glycosylating enzymes derived from cells which normally provide such processing, e.g., mammalian glycosylation enzymes. Deglycosylation enzymes are also contemplated.
  • a major group of derivatives are covalent conjugates of the DNAMl protein or fragments thereof with other proteins or polypeptides. These derivatives can be synthesized in recombinant culture such as N- or C- terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups. Preferred antigen derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues. Fusion polypeptides between the DNAMl proteins and other homologous or heterologous proteins are also provided.
  • Homologous polypeptides may be fusions between different surface markers, resulting in, e.g., a hybrid protein exhibiting receptor binding specificity.
  • heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins.
  • Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of an antigen, e.g., a receptor-binding segment, so that the presence or location of the fused antigen may be easily determined. See, e.g., Dull et al., U.S. Patent No. 4,859,609.
  • gene fusion partners include bacterial ⁇ - galactosidase, trpE, Protem A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating factor. See, e.g., Godowski et al. (1988) Science 241:812-816.
  • polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups.
  • the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands.
  • Fusion proteins will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods. Techniques for nucleic acid manipulation and expression are described generally, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed.), Vols. 1-3, Cold Spring Harbor Laboratory.
  • This invention also contemplates the use of derivatives of the DNAMl proteins other than variations in amino acid sequence or glycosylation.
  • Such derivatives may involve covalent or aggregative association with chemical moieties. These derivatives generally fall into the three classes: (1) salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, for example with cell membranes.
  • covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of antigens or other binding proteins.
  • a DNAMl antigen can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated
  • Sepharose by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of anti-DNAMl protein antibodies or its receptor or other binding partner.
  • the DNAMl antigens can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
  • Purification of DNAMl protein may be effected by immobilized antibodies or binding partners.
  • a solubilized DNAMl antigen or fragment of this invention can be used as an immunogen for the production of antisera or antibodies specific for the protein or fragments thereof.
  • the purified antigen can be used to screen monoclonal antibodies or binding fragments prepared by immunization with various forms of impure preparations containing the protein.
  • the term "antibodies” also encompasses antigen binding fragments of natural antibodies.
  • the purified DNAMl proteins can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of the protein or cell fragments containing the antigen, both of which may be diagnostic of an abnormal or specific physiological or disease condition. Additionally, antigen fragments may also serve as immunogens to produce the antibodies of the present invention, as described immediately below.
  • this invention contemplates antibodies raised against amino acid sequences encoded by nucleotide sequences shown in SEQ ID NO: 1, or fragments of proteins containing them.
  • this invention contemplates antibodies having binding affinity to or being raised against specific fragments which are predicted to lie outside of the lipid bilayer.
  • the invention also provides means to isolate a group of related antigens displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects of the antigens will be greatly accelerated by the isolation and characterization of distinct species variants.
  • the present invention provides useful probes for identifying additional homologous genetic entities in different species.
  • the isolated genes will allow transformation of cells lacking expression of a corresponding DNAMl protein, e.g., either species types or cells which lack corresponding antigens and should exhibit negative background activity. Expression of transformed genes will allow isolation of antigenically pure cell lines, with defined or single specie variants. This approach will allow for more sensitive detection and discrimination of the physiological effects of DNAMl proteins.
  • Subcellular fragments e.g., cytoplasts or membrane fragments, can be isolated and used.
  • functional domains or segments can be substituted between species variants or related proteins to determine what structural features are important in both binding partner affinity and specificity, as well as signal transduction.
  • An array of different variants will be used to screen for molecules exhibiting combined properties of interaction with different species variants of binding partners.
  • Antigen intemalization may occur under certain circumstances, and interaction between intracellular components and "extracellular" segments of proteins involved in interactions may occur.
  • the specific segments of interaction of DNAMl protein with other intracellular components may be identified by mutagenesis or direct biochemical means, e.g., cross-linking or affinity methods. Structural analysis by crystallographic or other physical methods will also be applicable. Further investigation of the mechanism of biological function will include study of associated components which may be isolatable by affinity methods or by genetic means, e.g., complementation analysis of mutants.
  • controlling elements associated with the antigens may exhibit differential developmental, tissue specific, or other expression patterns. Upstream or downstream genetic regions, e.g., control elements, are of interest.
  • the present invention provides important reagents related to antigen-binding partner interaction.
  • the foregoing description has focused primarily upon the human DNAMl protein, those of skill in the art will immediately recognize that the invention encompasses other closely related antigens, e.g., other primate species or allelic variants, as well as variants and other members of the family.
  • Antibodies can be raised to the various DNAMl proteins, including species or allelic variants, and fragments thereof, both in their naturally occurring forms and in their recombinant forms. Antibodies can be prepared against the polypeptides of the invention using standard methods. As used herein, the word "antibody” refers to both polyclonal and monoclonal antibodies. It also includes whole immunoglobulins and antigen binding fragments thereof. Additionally, antibodies can be raised to DNAMl proteins in either their active forms or in their inactive forms. Anti-idiotypic antibodies are also contemplated.
  • Antibodies, including binding fragments and single chain versions, against predetermined fragments of the antigens can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective DNAMl proteins, or screened for agonistic or antagonistic activity, e.g., mediated through a binding partner. These monoclonal antibodies will usually bind with a Kr) of better than about 1 mM, and preferably at least about 3 ⁇ M or better.
  • the antibodies, including antigen binding fragments, of this invention can have significant diagnostic or therapeutic value.
  • They can be potent antagonists that bind to a binding partner and inhibit antigen binding or inhibit the ability of an antigen to elicit a biological response. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides so that when the antibody binds to the antigen, a cell expressing it, e.g., on its surface, is killed. Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting.
  • the polyclonal antibodies can be produced by immunizing a host animal such as a rabbit, rat, goat, sheep, mouse, etc. with one of the polypeptides. Preferably, one or more booster injections are given after the initial injection, to increase the antibody titer. Blood is then drawn from the animal and serum is prepared and screened by standard methods such as enzyme-linked immunosorbent assay (ELISA) using the polypeptide as the antigen.
  • ELISA enzyme-linked immunosorbent assay
  • the immunogenicity of the polypeptides is increased by combination with an adjuvant and /or by conversion to a larger form prior to immunization.
  • Suitable adjuvants for the vaccination of animals include but are not limited to Adjuvant 65 (containing peanut oil, mannide monooleate and aluminum monostearate); Freund's complete or incomplete adjuvant; mineral gels such as aluminum hydroxide, aluminum phosphate and alum; surfactants such as hexadecylamine, octadecylamine, lysolecithin, dimethyldioctadecylammonium bromide, N,N-dioctadecyl-N',N'-bis(2-hydroxymethyl) propanediamine, methoxyhexadecylglycerol and pluronic polyols; polyanions such as pyran, dextran sulfate, poly IC, polyacrylic acid and carbopol; peptides such as muramyl dipeptide, dimethylglycine and tuftsin; and oil emulsions.
  • the polypeptides could also be administered following incorporation
  • the immunogenicity of the polypeptides can also be enhanced by cross-linking or by coupling to an immunogenic carrier molecule (i.e., a macromolecuie having the property of independently eliciting an immunological response in a host animal, to which the polypeptides of the invention can be covalently linked).
  • an immunogenic carrier molecule i.e., a macromolecuie having the property of independently eliciting an immunological response in a host animal, to which the polypeptides of the invention can be covalently linked.
  • Cross-linking or conjugation to a carrier molecule may be required because small polypeptides sometimes act as haptens (molecules which are capable of specifically binding to an antibody but incapable of eliciting antibody production, i.e., they are not immunogenic). Conjugation of such polypeptides to an immunogenic carrier molecule renders the fragments immunogenic through what is commonly known as the "carrier effect".
  • Suitable carrier molecules include, e.g., proteins and natural or synthetic polymeric compounds such as polypeptides, polysaccharides, lipopolysaccharides etc.
  • a useful carrier is a glycoside called Quil A. which has been described by Morein et al ., Nature 305:457 (1984).
  • Protein carrier molecules are especially preferred, including but not limited to keyhole limpet hemocyanin and mammalian serum proteins such as human or bovine gammaglobulin, human, bovine or rabbit serum albumin, or methylated or other derivatives of such proteins.
  • Other protein carriers will be apparent to those skilled in the art.
  • the protein carrier will be foreign to the host animal in which antibodies against the polypeptides are to be elicited.
  • Covalent coupling to the carrier molecule can be carried out using methods well known in the art, the exact choice of which will be dictated by the nature of the carrier molecule used.
  • the immunogenic carrier molecule is a protein
  • the polypeptides of the invention can be coupled, e.g., using water soluble carbodiimides such as dicyclohexylcarbodiimide or glutaraldehyde.
  • Coupling agents such as these can also be used to cross-link the polypeptides to themselves without the use of a separate carrier molecule. Such cross-linking into aggregates can also increase immunogenicity.
  • Serum produced from animals thus immunized can be used directly.
  • the IgG fraction can be separated from the serum using standard methods such as plasmaphoresis or adsorption chromatography using IgG specific adsorbents such as immobilized Protein A.
  • the antibodies of this invention can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for ability to bind to the antigens without inhibiting binding by a partner. As neutralizing antibodies, they can be useful in competitive binding assays. They will also be useful in detecting or quantifying DNAMl protein or its binding partners.
  • Antigen fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens.
  • An antigen and its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions. Dover Publications, New York, and Williams et al. (1967) Methods in Immunology and Immunochemistry. Vol. 1, Academic Press, New York, for descriptions of methods of preparing polyclonal antisera.
  • a typical method involves hyperimmunization of an animal with an antigen. The blood of the animal is then collected shortly after the repeated immunizations and the gamma globulin is isolated.
  • monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc.
  • Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites et al. (eds.) Basic and Clinical Immunology (4th ed.), Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice (2d ed.) Academic Press, New York; and particularly in Kohler and Milstein (1975) in Nature 256: 495-497, which discusses one method of generating monoclonal antibodies.
  • this method involves injecting an animal with an immunogen.
  • the animal is then sacrificed and cells taken from its spleen, which are then fused with myeloma cells.
  • the result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro.
  • the population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen.
  • the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also, recombinant immunoglobulins may be produced, see Cabilly, U.S. Patent No. 4,816,567. The antibodies of this invention can also be used for affinity chromatography in isolating the protein.
  • Columns can be prepared where the antibodies are linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate may be passed through the column, the column washed, followed by increasing concentrations of a mild denaturant, whereby the purified DNAMl protein will be released.
  • a solid support e.g., particles, such as agarose, Sephadex, or the like
  • the antibodies may also be used to screen expression libraries for particular expression products. Usually the antibodies used in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
  • Antibodies raised against each DNAMl protein will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens.
  • the present invention provides reagents which will find use in diagnostic applications as described elsewhere herein, e.g., in the general description for physiological or developmental abnormalities, or below in the description of kits for diagnosis.
  • This invention also provides reagents with significant therapeutic value.
  • the DNAMl protein naturally occurring or recombinant
  • fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to DNAMl protein, should be useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions; and abnormal cytotoxic activity.
  • Abnormal proliferation, regeneration, degeneration, and atrophy may be modulated by appropriate therapeutic treatment using the compositions provided herein.
  • a disease or disorder associated with abnormal expression or abnormal signaling by a DNAMl antigen should be a likely target for an agonist or antagonist of the protein.
  • Other abnormal developmental conditions are known in the cell types shown to possess DNAMl antigen mRNA by Northern blot analysis. See Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; and Thorn et al. Harrison's Principles of Intemal Medicine, McGraw-Hill, N.Y. These problems may be susceptible to prevention or treatment using compositions provided herein.
  • Recombinant antibodies which bind to DNAMl can be purified and then administered to a patient.
  • These reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients.
  • additional active or inert ingredients e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers and excipients.
  • These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations.
  • This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.
  • DNAMl for binding partners or compounds having binding affinity to DNAMl antigen
  • Screening using DNAMl for binding partners or compounds having binding affinity to DNAMl antigen can be performed, including isolation of associated components. Subsequent biological assays can then be utilized to determine if the compound has intrinsic biological activity and is therefore an agonist or antagonist in that it blocks an activity of the antigen.
  • This invention further contemplates the therapeutic use of antibodies to DNAMl protein as antagonists. This approach should be particularly useful with other DNAMl protein species variants and other members of the family.
  • reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics. 9th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn.
  • Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index. Merck & Co., Rahway, New Jersey. Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ⁇ M concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar), and most preferably less than about 1 fM (femtomolar), with an appropriate carrier. Slow release formulations, or a slow release apparatus will often be utilized for continuous administration. Typically, a lower effective dose is preferred.
  • DNAMl protein, fragments thereof, and antibodies to it or its fragments, antagonists, and agonists may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.
  • Therapeutic formulations may be administered in most any conventional dosage formulation. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. See, e.g., Gilman et al. (eds.) (1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and
  • the therapy of this invention may be combined with or used in association with other agents useful in modulating cytotoxicity, e.g., via the LFA- ICAM 1, 2, or 3 pathways, or the CD2:CD58 or CD48 pathways. See, e.g., Weiss (1994) in Paul (ed.)
  • Both the naturally occurring and the recombinant form of the DNAMl proteins of this invention are particularly useful in kits and assay methods which are capable of screening compounds for binding activity to the proteins.
  • kits and assay methods which are capable of screening compounds for binding activity to the proteins.
  • automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor et al. (1991) Science 251:767-773, which describes means for testing of binding affinity by a plurality of defined polymers synthesized on a solid substrate.
  • the development of suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble DNAMl protein as provided by this invention.
  • This invention is particularly useful for screening compounds by using recombinant antigen in any of a variety of drug screening techniques.
  • the advantages of using a recombinant protein in screening for specific ligands include: (a) improved renewable source of the antigen from a specific source; (b) potentially greater number of antigen molecules per cell giving better signal to noise ratio in assays; and (c) species variant specificity (theoretically giving greater biological and disease specificity).
  • the purified protein may be tested in numberous assays, typically in vitro assays, which evaluate biologically relevant responses. See, e.g., Coligan Current Protocols in Immunology: Hood et al.
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing the DNAMl antigens.
  • Cells may be isolated which express an antigen in isolation from other functionally equivalent antigens.
  • Such cells either in viable or fixed form, can be used for standard protein-protein binding assays. See also, Parce et al. (1989) Science 246:243-247; and Owicki et al. (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitive methods to detect cellular responses.
  • DNAMl protein mediated functions e.g., second messenger levels, i.e., Ca++; cell proliferation; inositol phosphate pool changes; and others.
  • Some detection methods allow for elimination of a separation step, e.g., a proximity sensitive detection system.
  • Calcium sensitive dyes will be useful for detecting Ca ++ levels, with a fluorimeter or a fluorescence cell sorting apparatus.
  • Another method utilizes membranes from transformed eukaryotic or prokaryotic host cells as the source of the DNAMl protein. These cells are stably transformed with DNA vectors directing the expression of a membrane associated DNAMl protein, e.g., an engineered membrane bound form. Essentially, the membranes would be prepared from the cells and used in any receptor/ligand type binding assay such as the competitive assay set forth above.
  • Still another approach is to use solubilized, unpurified or solubilized, purified DNAMl protein from transformed eukaryotic or prokaryotic host cells. This allows for a "molecular" binding assay with the advantages of increased specificity, the ability to automate, and high drug test throughput.
  • Another technique for drug screening involves an approach which provides high throughput screening for compounds having - 37 - suitable binding affinity to DNAMl and is described in detail in Geysen, European Patent Application 84/03564, published on September 13, 1984.
  • a solid substrate e.g., plastic pins or some other appropriate surface, see Fodor et al. (1991).
  • all the pins are reacted with solubilized, unpurified or solubilized, purified DNAMl binding composition, and washed.
  • the next step involves detecting bound binding composition.
  • Rational drug design may also be based upon structural studies of the molecular shapes of the DNAMl protein and other effectors or analogues. Effectors may be other proteins which mediate other functions in response to antigen binding, or other proteins which normally interact with the antigen, e.g., DNAMl ligand.
  • One means for determining which sites interact with specific other proteins is a physical structure determination, e.g., x-ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions.
  • x-ray crystallography or 2 dimensional NMR techniques.
  • Purified DNAMl protein can be coated directly onto plates for use in the aforementioned drug screening techniques. However, non- neutralizing antibodies to these ligands can be used as capture antibodies to immobilize the respective ligand on the solid phase.
  • This invention also contemplates use of DNAMl proteins, fragments thereof, peptides, and their fusion products in a variety of diagnostic kits and methods for detecting the presence of a binding composition.
  • the kit will have a compartment containing either a defined DNAMl peptide or gene segment or a reagent which recognizes one or the other, e.g., antigen fragments or antibodies.
  • a kit for determining the binding affinity of a test compound to a DNAMl protein would typically comprise a test compound; a labeled compound, for example an antibody having known binding affinity for the antigen; a source of DNAMl protein (naturally occurring or recombinant); and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the antigen.
  • a test compound for example an antibody having known binding affinity for the antigen
  • a source of DNAMl protein naturally occurring or recombinant
  • a means for separating bound from free labeled compound such as a solid phase for immobilizing the antigen.
  • a preferred kit for determining the concentration of, for example, a DNAMl protein in a sample would typically comprise a labeled compound, e.g., antibody, having known binding affinity for the antigen, a source of antigen (naturally occurring or recombinant) and a means for separating the bound from free labeled compound, for example, a solid phase for immobilizing the DNAMl protein. Compartments containing reagents, and instructions, will normally be provided.
  • One method for determining the concentration of DNAMl protein in a sample would typically comprise the steps of: (1) preparing membranes from a sample comprised of a membrane bound DNAMl protein source; (2) washing the membranes and suspending them in a buffer; (3) solubilizing the antigen by incubating the membranes in a culture medium to which a suitable detergent has been added; (4) adjusting the detergent concentration of the solubilized antigen; (5) contacting and incubating said dilution with radiolabeled antibody to form complexes; (6) recovering the complexes such as by filtration through polyethyleneimine treated filters; and (7) measuring the radioactivity of the recovered complexes.
  • Antibodies including antigen binding fragments, specific for the DNAMl protein or fragments are useful in diagnostic applications to detect the presence of elevated levels of DNAMl protein and /or its fragments.
  • diagnostic assays can employ lysates, live cells, fixed cells, immunofiuorescence, cell cultures, body fluids, and further can involve the detection of antigens related to the protein in serum, or the like. Diagnostic assays may be homogeneous (without a separation step between free reagent and protein-protein complex) or heterogeneous (with a separation step).
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • EMIT enzyme-multiplied immunoassay technique
  • SFIA substrate-labeled fluorescent immunoassay
  • uniabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to a DNAMl protein or to a particular fragment thereof.
  • Similar assays have also been extensively discussed in the literature. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH.
  • Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against a DNAMl protein, as such may be diagnostic of various abnormal states. For example, overproduction of DNAMl protein may result in production of various immunological reactions which may be diagnostic of abnormal physiological states, particularly in proliferative cell conditions such as cancer or abnormal differentiation.
  • the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay.
  • the protocol, and the label either labeled or uniabeled antibody, or labeled DNAMl protein is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like.
  • the kit will also contain instructions for proper use and disposal of the contents after use.
  • the kit has compartments for each useful reagent.
  • the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
  • labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.
  • the antigen, test compound, DNAMl protein, or antibodies thereto can be labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups: radiolabels such as 125 ⁇ enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization.
  • Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups.
  • the DNAMl protein can be immobilized on various matrixes followed by washing. Suitable matrixes include plastic such as an ELISA plate, filters, and beads. Methods of immobilizing the DNAMl protein to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin- avidin.
  • the last step in this approach involves the precipitation of protein-protein complex by any of several methods including those utilizing, e.g., an organic solvent such as polyethylene glycol or a salt such as ammonium sulfate.
  • oligonucleotide or polynucleotide sequences taken from the sequence of a DNAMl protein. These sequences can be used as probes for detecting levels of antigen message in samples from patients suspected of having an abnormal condition, e.g., cancer or developmental problem.
  • an abnormal condition e.g., cancer or developmental problem.
  • the preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature.
  • an oligonucleotide probe should have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes may be up to several kilobases.
  • Various labels may be employed, most commonly radionuclides, particularly 32p. However, other techniques may also be employed, such as using biotin modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • probes to the novel anti-sense RNA may be carried out in any conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT), and hybrid arrested translation (HART). This also includes amplification techniques such as polymerase chain reaction (PCR).
  • kits which also test for the qualitative or quantitative presence of other markers are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers. Thus, kits may test for combinations of markers. See, e.g., Viallet et al. (1989) Progress in Growth Factor Res. 1:89-97.
  • the DNAMl protein should interact with a ligand based, e.g., upon its similarity in structure and function to other cell markers exhibiting developmental and cell type specificity of expression. Methods to isolate a ligand are made available by the ability to make purified DNAMl for screening programs. Soluble or other constructs using the DNAMl sequences provided herein will allow for screening or isolation of DNAMl specific ligands.
  • Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel et al. (1987 and periodic supplements); Deutscher (1990) "Guide to Protein Purification” in Methods in Enzymology. vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA. Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence. See, e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990)
  • mice were immunized by standard protocols with a cytotoxic human NK cell clone. After repeated immunizations, splenocytes were fused with the Sp2/0 fusion partner and hybridomas were selected in growth medium containing azaserine by standard procedures. The supematants from individual hybridomas were screened in a redirected killing assay against the Fc receptor bearing tumor cell line, P815. This particular assay allows for the detection of monoclonal antibodies against signal transducing surface molecules expressed on NK cells and results in the cytolysis of the P815 tumor cell line. The original anti-DNAM-1 monoclonal antibody, designated DXll was identified using this assay. DXll is a monoclonal antibody of the IgGl,K isotype.
  • the binding reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods. Supematants can be screened for binding to the DNAMl antigen.
  • the DNAMl antigen may be either purified by methods provided herein, or may be recombinantly expressed on a cell.
  • DNAMl was determined to be expressed prominently on a majority of peripheral blood NK cells, T cells, monocytes and a subset of B cells. DNAMl was not expressed on granulocytes, platelets or red blood cells. See, e.g., Table 1. In the thymus, DNAMl is expressed on the most mature thymocytes coexpressing high surface density CD3. DNAMl is expressed on a variety of transformed hematopoietic cell lines, however, it is not generally expressed on epithelial, neuronal, or fibroblast cell lines. The distribution of DNAMl mRNA may be determined by PCR or hybridization techniques, although the amounts of DNAMl message are extremely low. This feature accounted for enormous difficulty in isolating a nucleic acid clone, as standard techniques had failed.
  • a recombinant DNAMl construct is prepared which is fused to a useful affinity reagent, e.g., FLAG peptide, useful for purifying the expression product of the construct.
  • a useful affinity reagent e.g., FLAG peptide
  • the sequence allows for efficient affinity purification of the soluble product. Appropriate secretion or processing sites may also be engineered into the construct by standard methods. Purification is achieved by use of affinity purification, e.g., antibodies against the antigen, or by standard protein purification methods. Typically, the affinity reagents or purification procedures can be performed using recombinant receptor.
  • the DNAMl protein is isolated by a combination of affinity chromatography using the DNAMl specific binding compositions, e.g., antibody, as a specific binding reagent in combination with protein purification techniques allowing separation from other proteins and contaminants. Similar techniques using human cell assays and human cell sources are applied to isolate a human antigen. V. Isolation of a DNA clone encoding DNAMl protein.
  • DNAMl protein was then purified to near homogeneity from peripheral blood mononuclear cells by standard protein purification techniques, including both lectin affinity and antibody affinity chromatography.
  • the purified DNAMl protein was then subjected to peptide sequencing, thus obtaining both N-terminal and internal amino acid fragments.
  • Degenerate oligonucleotides were generated based on the amino acid fragments and PCR techniques were applied to isolate a short fragment of DNA encoding a small portion of the gene. This small fragment of the DNAMl gene was then used as a probe to isolate the full length DNAMl gene.
  • the full length DNAMl gene is 2.6 kB with an open reading frame of 1008 nucleotides, which encodes 336 amino acids.
  • the DNAMl gene is a member of the immunoglobulin supergene family with two extracellular domains, a 26 amino acid transmembrane region, and a 61 amino acid cytoplasmic domain containing three potential tyrosine phosphorylation sites.
  • DNAMl is a signal transducing molecule expressed on NK cells and T cells.
  • cytotoxicity of various tumor cell lines is only partially inhibited by the DXll. If the LFA-1 /ICAMs and/or CD2/LFA-3 accessory pathways are blocked by antibodies in addition to DNAMl, however, the cytotoxicity against these tumor cell lines is also completely inhibited. See, e.g., Table 5. Table 5. Cytotoxicity of various tumor cell lines by freshly isolated NK cells, NK cell clones, T cell clones, and antigen specific CTLs (AS547 and JS86) are inhibited by the anti-DNAMl monoclonal antibody DXll alone or in combmation with antibodies against CDI 8 and CD2 adhesion pathways
  • DNAMl molecule is an important accessory molecule employed by NK cells and T cells during the cytolysis of a variety of tumor cell targets. It may suggest that modulation with a DNAMl signal in combination with either LFA-1/ICAM or CD2/LFA-3 may provide more efficient killing or suppression of killing.
  • NK cells Incubation of NK cells with the anti-DNAMl monoclonal antibody, DXll, and cross linking by a secondary antibody (goat anti-mouse lg, GaMIg) induces strong lymphokine secretion from NK cells. High concentrations of IFN-g, TNF-a, and GM-CSF are secreted from NK cells when the DNAMl molecules are engaged by crosslinked DXll monoclonal antibody. See Table 6.
  • IFN-g (pg/ml) TNF-a (pg/ml) GM-CSF (ng/ml) media only 160.0 15.0 2.40
  • the DNAMl is used for screening of an expression library made from a cell line which expresses a DNAMl binding protein, e.g., a ligand.
  • Standard staining techniques are used to detect or sort intracellular or surface expressed ligand, or surface expressing transformed cells are screened by panning. Screening of intracellular expression is performed by various staining or immunofiuorescence procedures. See, e.g., McMahan et al. (1991) EMBO T. 10:2821-2832.
  • HBSS Hank's Buffered Saline Solution
  • PFA paraformaldehyde
  • glucose glucose
  • the slides may be stored at -80° C after all liquid is removed.
  • 0.5 ml incubations are performed as follows. Add HBSS/saponin (0.1%) with 32 ml/ml of IM NaN3 for 20 min. Cells are then washed with HBSS/saponin IX. Soluble antibody is added to cells and incubate for 30 min. Wash cells twice with HBSS/saponin.
  • Second antibody e.g., Vector anti-mouse antibody
  • second antibody e.g., Vector anti-mouse antibody
  • Prepare ELISA solution e.g., Vector Elite ABC horseradish peroxidase solution, and preincubate for 30 min.
  • Use e.g., 1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 ml HBSS/saponin.
  • ABC HRP solution and incubate for 30 min. Wash cells twice with HBSS, second wash for 2 min, which closes cells.
  • DAB Vector diaminobenzoic acid
  • DNAMl proteins are used to affinity purify or sort out cells expressing the ligand. See, e.g., Sambrook et al. or Ausubel et al.
  • AAG CCC TAT GCT GAG AGG GTT TAC TTT TTG AAT TCA ACG ATG GCT TCC 472 Lys Pro Tyr Ala Glu Arg Val Tyr Phe Leu Asn Ser Thr Met Ala Ser 75 80 85
  • CAG CCC CGT CAG ATC GAC CTC TTA ACT TAC TGC AAC TTG GTC CAT GGC 760 Gin Pro Arg Gin He Asp Leu Leu Thr Tyr Cys Asn Leu Val His Gly 170 175 180

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Abstract

Cette invention concerne un antigène de surface de lymphocyte T et de cellule tueuse naturelle (TN) qui provient d'un mammifère, des réactifs liés à ce dernier et comprenant des protéines purifiées, des anticorps spécifiques et des acides nucléiques codant ledit antigène. Des procédés d'utilisation de ces réactifs et des nécessaires de diagnostic sont également décrits.
PCT/US1996/018551 1995-11-30 1996-11-26 Molecule d'adn, antigene tn et molecule d'adhesion appartenant a la superfamille des immunoglobulines WO1997020046A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006557A2 (fr) * 1997-08-01 1999-02-11 Schering Corporation Proteines de membrane cellulaire mammalienne et reactifs associes
US6416973B1 (en) 1997-08-01 2002-07-09 Schering Corporation Nucleic acids encoding mammalian cell membrane protein MDL-1

Citations (2)

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Publication number Priority date Publication date Assignee Title
WO1992001049A2 (fr) * 1990-07-13 1992-01-23 The General Hospital Corporation Antigene de surface de cellule cd53 et son utilisation
US5506126A (en) * 1988-02-25 1996-04-09 The General Hospital Corporation Rapid immunoselection cloning method

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Publication number Priority date Publication date Assignee Title
US5506126A (en) * 1988-02-25 1996-04-09 The General Hospital Corporation Rapid immunoselection cloning method
WO1992001049A2 (fr) * 1990-07-13 1992-01-23 The General Hospital Corporation Antigene de surface de cellule cd53 et son utilisation

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Title
SHIBUYA A ET AL: "DNAM-1, a novel adhesion molecule involved in the cytolytic function of T lymphocytes.", IMMUNITY, JUN 1996, 4 (6) P573-81, UNITED STATES, XP000618600 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999006557A2 (fr) * 1997-08-01 1999-02-11 Schering Corporation Proteines de membrane cellulaire mammalienne et reactifs associes
WO1999006557A3 (fr) * 1997-08-01 1999-05-20 Schering Corp Proteines de membrane cellulaire mammalienne et reactifs associes
US6416973B1 (en) 1997-08-01 2002-07-09 Schering Corporation Nucleic acids encoding mammalian cell membrane protein MDL-1
US6953843B2 (en) 1997-08-01 2005-10-11 Schering Corporation Antibodies and fragments thereof to MDL-1 protein
EP1798287A3 (fr) * 1997-08-01 2007-11-14 Schering Corporation Protéines de membrane cellulaire mammalienne et réactifs associes
EP1798286A3 (fr) * 1997-08-01 2007-11-14 Schering Corporation Protéines de membrane cellulaire mammalienne et réactifs associes
US7319140B2 (en) 1997-08-01 2008-01-15 Schering Corporation Binding compounds to DAP12 proteins
US7659093B2 (en) 1997-08-01 2010-02-09 Schering Corporation DAP12 nucleic acids
JP2010099076A (ja) * 1997-08-01 2010-05-06 Schering Corp 哺乳動物細胞膜タンパク質；関連試薬

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